The long-term objective of this proposal is to characterize the role of endothelins (ETs) in regulating reproductive hormone secretion. The aims of this proposal revolve around elucidation of the mechanisms whereby ETs' affect pituitary prolactin (PRL) secretion. It is submitted under the area of Reproductive Endocrinology in response to the Special Emphasis Area solicitation of the NICHD. The first specific aim of this proposal is to characterize the physiological role of endothelins in the control of lactotroph function during the estrous cycle. This will be accomplished by examining the effects of specific ET antagonists on PRL secretion in vivo. The second specific aim is to characterize the autocrine and paracrine actions of ETs in controlling PRL secretion. This will be assessed in vitro (a) at the single cell level by using reverse hemolytic plaque assay methods on pituitary cells collected during different stages of the estrous cycle and (b) in a multicellular environment by using pituitary cell micro-aggregate cultures in a cell-perfusion system. The effects of ET receptor antagonists on the secretory activity of lactotrophs will be tested in basal and secretagogue-induced conditions. The third specific aim is to identify signal transduction mechanisms by which ETs affect PRL secretion. We will use dispersed pituitary cells enriched in lactotrophs and/or an established pituitary cell line of lactotroph origin expressing ETA receptors. We will employ specific inhibitors of protein kinase cascades thought to be involved in the regulation of PRL secretion. Specific emphasis will be made on investigating the role of protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) dependent signaling pathways. The fourth specific aim is to establish the ionic basis for the effects of endothelins on pituitary lactotrophs. In these studies we will seek to confirm our hypothesis that ET-induced multiple signaling pathways converge upon a unique population of BKCa channels and that modulation of these channels is responsible for most of the effects of ETs on PRL secretion. Using cultured lactotrophs, we will employ patch clamp methods in cell-attached and inside-out configuration to gain information on gating kinetics at the single channel level. The fifth specific aim is to establish the mechanism of cross-talk between dopaminergic D2 and endothelin ETA receptors. The investigation will focus on the mechanisms by which dopamine transforms the response of lactotrophs to ET from inhibitory to stimulatory. These studies will provide, for the first time, a thorough characterization of the physiological role of ETs in regulating pituitary hormone secretion. Accomplishment of these specific aims will clarify a heretofore undescribed cellular mechanism regulating PRL secretion and suggest novel therapeutic approaches to control secretory functions of the hypothalamo-pituitary axis.